Chinese cabbage BcpFLC3 protein nucleotide sequence
A nucleotide sequence and Chinese cabbage technology, applied in the field of genetic engineering, can solve problems that have not yet been discovered
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Embodiment 1
[0124] Cloning of Bolting Resistance Gene in Chinese Cabbage
[0125] 1. Tissue separation (isolation)
[0126] Chinese cabbage (the variety is "Spring King") was purchased from the market, and the Chinese cabbage was placed at 28°C to germinate for 24 hours, and then sowed in the greenhouse. When the Chinese cabbage leaves were 4-5 pieces, DNA or RNA was prepared to be extracted.
[0127] 2. RNA isolation (RNA isolation)
[0128] Take part of the tissue, grind it with a mortar, add it to a 1.5mL EP tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to 1.5mL EP tube, and extract total RNA (TRIzol Reagents, GIBCO BRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.
[0129] 3. Cloning of Full-length cDNA
[0130] According to the amino acid conserved sequence of the Arabidopsis flowering inhibitory ge...
Embodiment 2
[0142] Sequence information and homology analysis of genes related to bolting resistance in Chinese cabbage
[0143] The length of the novel Chinese cabbage anti-bolting full-length cDNA is 851 bp, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at 55-645 nucleotides (591 nucleotides). According to the full-length cDNA, the anti-bolting amino acid sequence of Chinese cabbage was deduced, with a total of 197 amino acid residues, a molecular weight of 21644.98 Daltons, and an isoelectric point (pI) of 9.25. See SEQ ID NO.3 for the detailed sequence.
[0144] The full-length cDNA sequence and its encoded protein related to bolting resistance of Chinese cabbage were nucleotide aligned in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases using BLAST program. Protein homology search, it was found that it has 98% identity with Brassica napus flowering suppressor gene MA...
Embodiment 3
[0146] Eukaryotic expression of Chinese cabbage anti-bolting-related protein or polypeptide in Arabidopsis and identification of bolting of transgenic plants
[0147] Construction of expression vector containing target gene (Chinese cabbage BcpFLC3 gene)
[0148] According to the full-length sequence (SEQ ID NO.3) related to anti-bolting of Chinese cabbage, design primers to amplify the complete coding reading frame, and introduce restriction endonuclease sites on the upstream and downstream primers respectively (this can be selected depending on the vector) in order to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Chinese cabbage anti-bolting gene cDNA was cloned into an intermediate vector (such as pBluescript), and further cloned into a binary expression vector (such as pBI121 and improved pCAMBIA2300) , under the premise of ensuring the correct reading frame, the identified expression vector ...
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